Primaquine dipeptide derivatives bearing an imidazolidin-4-one moiety at the N-terminus as potential antimalarial prodrugs

Article abstract of DOI:10.1016/j.ejmech.2009.01.018

Primaquine dipeptide derivatives bearing an imidazolidin-4-one moiety at the N-terminus were synthesized and evaluated as potential transmission-blocking antimalarial prodrugs. All compounds were hydrolyzed to the parent dipeptide derivative of primaquine in neutral and basic solutions, with half lives ranging from 0.7 to 31 h at 37 °C, depending on the nature of the substituents present in the imidazolidin-4-one moiety and in the C-terminal amino acid directly coupled to primaquine. The antimalarial activity was studied for selected compounds using a model consisting of Plasmodium berghei, BalbC mice and Anopheles stephensi mosquitoes. The imidazolidin-4-one derived from Ala-Ala-primaquine and acetone reduced the transmission of the infection to mosquitoes more efficiently than primaquine as shown by the significant decrease in the number of oocysts in the midguts of the mosquitoes at 10 and 50 μmol/kg when compared to the control.

Full text of DOI:10.1016/j.ejmech.2009.01.018

European Journal of Medicinal Chemistry 44 (2009) 2506–2516
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Primaquine dipeptide derivatives bearing an imidazolidin-4-one moiety
at the N-terminus as potential antimalarial prodrugs
a
b
b
a
,
c,
**
*
´
´
´
Nuno Vale , Fatima Nogueira , Virgılio E. do Rosario , Paula Gomes , Rui Moreira
a
´
ˆ
CIQUP, Departamento de Quımica, Faculdade de Ciencias, Universidade do Porto, R. Campo Alegre, P-4169-007 Porto, Portugal
b CMDT-LA, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, R. Junqueira 96, P- 1349-008 Lisboa, Portugal
c
´
iMed.UL, Faculdade de Farmacia, Universidade de Lisboa, Av. Professor Gama Pinto, P-1649-003 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Primaquine dipeptide derivatives bearing an imidazolidin-4-one moiety at the N-terminus were
synthesized and evaluated as potential transmission-blocking antimalarial prodrugs. All compounds
were hydrolyzed to the parent dipeptide derivative of primaquine in neutral and basic solutions, with
half lives ranging from 0.7 to 31 h at 37 ꢀC, depending on the nature of the substituents present in the
imidazolidin-4-one moiety and in the C-terminal amino acid directly coupled to primaquine. The anti-
malarial activity was studied for selected compounds using a model consisting of Plasmodium berghei,
BalbC mice and Anopheles stephensi mosquitoes. The imidazolidin-4-one derived from Ala-Ala–prima-
quine and acetone reduced the transmission of the infection to mosquitoes more efficiently than pri-
maquine as shown by the significant decrease in the number of oocysts in the midguts of the mosquitoes
Received 19 November 2008
Received in revised form
15 January 2009
Accepted 20 January 2009
Available online 29 January 2009
Keywords:
Imidazolidin-4-ones
Prodrugs
at 10 and 50 mmol/kg when compared to the control.
Primaquine
Malaria
Gametocytocidal activity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Peptide and amino acid derivatives of primaquine have been
prepared to reduce toxicity of the parent drug as well as to suppress
the metabolic pathway leading to 2 [15–18]. Despite the improved
activity/toxicity ratio, most of these derivatives are rapidly hydro-
lyzed to primaquine by aminopeptidases and endopeptidases [16,18],
suggesting that they might undergo extensive hydrolysis to the
parent drug in the intestinal lumen when given orally. Imidazolidin-
4-one formation was introduced as a useful prodrug approach to
protect the N-terminal amino acid residue of di-, tri- and pentapep-
tides against aminopeptidase-catalyzed hydrolysis [19–22]. The
synthetic approaches for the preparation of imidazolidin-4-ones
involve the reaction of the peptide with an aldehyde or ketone fol-
lowed by intramolecular cyclization. Recently, we prepared imida-
zolidin-4-ones, 3 (Scheme 1), as potential peptidase-stable prodrugs
of amino acid derivatives of primaquine [23–25]. Compounds 3 dis-
played gametocytocidal activity against Plasmodium berghei compa-
rable to that of primaquine, as well as antiplasmodial activity against
Plasmodium falciparum, but presented low aqueous solubility and,
unexpectedly, high stability in pH 7.4 buffer (half lives for hydrolysis
typically higher than 12 h), i.e. they cannot be considered as prodrugs
[24,26]. To overcome the problem of poor aqueous solubility dis-
played by compounds 3, we decided to incorporate the imidazolidin-
4-one moiety at the C-terminus of a dipeptide backbone coupled to
theparentdrug, e.g.4(Scheme 1).However, theseimidazolidin-4-one
peptidomimetics are extremely stable, both in human plasma and in
pH 7.4 buffer, as a result of N1-acylation [27]. The hydrolysis of
Primaquine, 1 (Scheme 1), is the only currently available drug
active against both the latent liver forms of the relapsing malaria
caused by Plasmodium vivax and Plasmodium ovale and the
gametocytes from all species of parasite causing human malaria
[1,2]. This 8-aminoquinoline is also active against Pneumocystis
carinii [3], Leishmania tropica [4] and Tripanosoma cruzi [5], the
causing agents of Pneumocystis pneumonia (PCP), visceral
leishmaniosis and Chagas’s disease, respectively. However, pri-
maquine presents a short plasma half-life (ca. 6 h) [6,7], as
a result of its rapid oxidative deamination to the inactive
metabolite carboxyprimaquine
2 (Scheme 1) [8–12]. Blood
toxicity, in particular the ability of primaquine to induce oxida-
tion of oxyhemoglobin to methemoglobin, has been also
a source of concern [13]. Primaquine causes haemolytic anemia
in patients with glucose-6-phosphate dehydrogenase (G6PD)
deficiency [12]. In addition, a recent report revealed that pri-
maquine may also affect gene expression in liver when given in
high doses and produce undesirable outcomes if consumed for
long periods [14].
* Corresponding author. Tel.: þ351 220402563; fax: þ351 220402659.
** Corresponding author. Tel.: þ351 217946477; fax: þ351 217946470.
E-mail addresses: pgomes@fc.up.pt (P. Gomes), rmoreira@ff.ul.pt (R. Moreira).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.018

N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
2507
MeO
N
HN
R
Me
O
O
R1
R1
R4
N
N
1, R = CH2NH2
2, R = CO2H
3, R =
4, R =
N
NH
R2
R2
NH2
R3
R3
O
Scheme 1.
imidazolidin-4-ones in neutral and basic media occurs via an SN1-
type mechanism [20,26] which involves the departure of an amide
leaving group and formation of an iminium ion at N1. Thus, removing
the nucleophilicity of the N1-nitrogen atom by acylation virtually
turns compounds 4 unreactive. With the objective of developing
prodrugs capable of reverting to the parent dipeptidyl primaquine
with half lives adequate for oral administration, we decided to
incorporate the imidazolidin-4-one moiety in the N-terminus of the
dipeptide backbone, e.g. 5 (Scheme 2). We now report the synthesis,
evaluation of the chemicalreactivityand antimalarial activityofnovel
imidazolidin-4-one derivatives of primaquine, 5 (Table 1).
chiral purity of amino acids, peptides and other amino acid deriv-
atives. In fact, it is well established that in what concerns chiral
stability of their Ca, amino acids and their derivatives are fairly
insensitive to acids [28,29].
Yields for the cyclization step range from reasonable to good and
seem to be dependent on the nature of the R1 substituent present in
intermediates 9. For example, the cyclization yield is significantly
lower when a bulky amino acid such as Leu or Phe is present as the
C-terminal residue (i.e. R1) of the dipeptide backbone than when is
present as the N-terminal residue (i.e. R4) (e.g. 5h versus 5d and 5l
versus 5n). This effect might reflect the steric requirements for the
cyclization of imine, 10 (Scheme 3), formed from the reaction of the
amino group in 9 with the appropriate ketone [23,30,31]. It can be
seen that conformation A (Scheme 3) of the imine 10 presents some
repulsion between the R1 group and the R2 and R3 substituents,
thus favouring reactions in which R1 ¼ H. Bulkier R4 substituents
might also favour cyclization of the imine, as they force the amide
nitrogen atom to get in closer proximity to the imine carbon atom.
For example, 5d (R1 ¼ H, R4 ¼ Bui) was formed in 75% yield, while
5a (R1 ¼ R4 ¼ H) was formed only in 41% yield. Similar observations
can be made for 5f versus 5g and for 5o versus 5p.
2. Chemistry
The synthesis of imidazolidin-4-one prodrugs 5 is illustrated in
Scheme 2. Briefly, primaquine reacted with the appropriate Na-Boc-
protected amino acid (BocAA1OH) using HOBt/DIPCI or HOBt/DCCI
methods. The corresponding Na-Boc-protected primaquine amino
acid amides (6a–d) were isolated, Boc removed with neat tri-
fluoroacetic acid (TFA) and the resulting trifluoroacetates were
converted into the corresponding free bases, 7a–d, with sodium
carbonate. Compounds 7 were reacted with the second Na-Boc-
protected amino acid (BocAA4OH) in the presence of triethylamine
(TEA) and HOBt/DIPCI or HOBt/DCCI, to yield the protected prima-
quine dipeptide amides 8a–i. These were converted to the depro-
tected products (9a–i) again with neat TFA followed by treatment
with sodium carbonate. Finally, compounds 9 were converted into
the corresponding imidazolidin-4-ones, 5a–s (Table 1), by reaction
with the appropriate symmetric ketone.
3. Results and discussion
3.1. Kinetics and products of hydrolysis
As revealed by HPLC analysis, compounds 5 hydrolyze quanti-
tatively at 37 ꢀC to the corresponding dipeptide derivative 9 over
the pH range 0.5–12. Products 9 were stable in these conditions and
in the time-scale of the decomposition of 5. The rates of hydrolyses
of imidazolidin-4-ones 5 at a fixed pH value were found to be
independent of buffer concentration over a 10-fold buffer concen-
tration range, indicating the absence of general acid or base catal-
ysis (e.g. Table 2). The influence of pH on the rate of hydrolysis is
shown in Fig. 1 for compounds 5a and 5s, where the logarithm of
the observed pseudo-first-order rate constant, kobs, is plotted
against pH. The pH–rate profiles for compounds 5 have a sigmoid
shape, with two pH-independent regions. Such sigmoid pH–rate
profile have been reported for other imidazolidin-4-ones [19,32] as
well as for their acyclic counterparts, N-Mannich bases [33], and
can be accounted for by assuming that both the protonated, SHþ,
and the unionized, S, forms of the substrate undergo spontaneous
hydrolysis (Scheme 4). The best computer fit (solid line) to the
experimental data for 5a and 5s in Fig. 1 was achieved using Eq. (1):
The structure of imidazolidin-4-ones 5 follows from their
spectroscopic data, which reveal the presence of two diastereomers
resulting from using racemic primaquine as starting material. For
example, the imidazolidin-4-one C-2 methyl proton signals of 5d
and 5e clearly appear as two sets of two singlets with overall
1:1:1:1 integration. Similarly, the two methyl groups at C-2 are
often visible as four signals in the 13C NMR spectra. In addition, the
1H NMR signal of the methylene protons on the glycine residue
linked to primaquine in 5b–e, 5m–n and 5q–s reveals the diaster-
eotopic nature of the two protons, which appear as an AB system at
d
ca. 4 ppm, with 2J ca. 15 Hz. Similar observations were recently
reported for the analogous peptidomimetic imidazolidin-4-ones 4
[27]. It should be outlined that identical spectroscopic behavior had
already been found for the Boc-protected precursors 6, whose
conversion into compounds 5 is carried out by standard Na-Boc-
amino acid acidolytic deprotection methods known to preserve the

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N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
MeO
MeO
(iii)
(i)
R4
N
O
N
O
1
H
N
HN
NHX
HN
NH
NH
NHX
Me
R1
Me
R1
O
6, X = BOC
7, X = H
8, X = BOC
(ii)
(ii)
9
, X = H
6a 7a
,
, R1 = H
8a,9a, R1 = H, R2 = H
6b,7b, R1 = Me,
6c,7c, R1 = Bui,
,
, R1 = H, R2 = Me
8b 9b
8c,9c, R1 = H, R2 = Pri
, R1 = CH2Ph
6d 7d
,
8d,9d, R1 = H, R2 = Bui
1
,
, R = H, R2 = CH2Ph
8e 9e
8f,9f, R1 = Me, R2 = Me
8g,9g, R1 = Me, R2 = (CH2)2SMe
,
, R1 = Bui, R2 = H
8h 9h
8i,9i, R1 = Me, R2 = H
8j,9j, R1 = CH2Ph, R2 = H
(iv)
MeO
R4
O
N
O
NH
R3
HN
N
N
H
R2
R1
Me
5
R1, R4 = H, Me, Pri, Bui, CH2Ph
R2 = R3 = Me, (CH2)4, (CH2)5, (CH2)6,
(CH2)2CH(CH3)(CH2)2
Scheme 2. Legend: (i) BocAA1OH, DCCI or DIPCI, HOBt, TEA, DCM, 0 ꢀC to r.t.; (ii) neat TFA, aq. 30% Na2CO3, r.t.; (iii) 7, BocAA4OH, DCCI or DIPCI, HOBt, TEA, DCM, 0 ꢀC to r.t.; (iv) 9,
R2COR3, TEA, MeOH, reflux.
h
i
constant for the neutral prodrug, kneut (Scheme 4), has a small
dependence on the size of the amino acid substituent R4 at the imi-
dazolidin-4-one C-5. For example, the order of half lives for the AA–
Gly–PQ series derived from acetone is Gly-Gly–PQ, 5a > Ala-Gly–PQ,
5b z Phe-Gly–PQ, 5e > Val-Gly–PQ, 5cwith5abeingonlyca.10times
more reactive than 5c. The exception is compound 5d, derived from
Leu-Gly–PQ and acetone, which is more reactive than expected based
on the size of the Leu side chain, which might reflect the importance
of polar effects on reactivity. The decrease of kneut with the size of the
amino acid substituent R4 at the imidazolidin-4-one C-5 is also
observable for the cyclohexanone series: Gly-Gly–PQ, 5i > Ala-Gly–
PQ, 5m > Phe-Gly–PQ, 5n. These results are consistent with those
reported for imidazolidin-4-ones based on amino acid derivatives of
primaquine and for imidazolidin-4-ones derived from dipeptides
[20,26] and can be ascribed to unfavourable steric interactions
between the amino acid substituent and the R2 and R3 substituents in
the iminium ion (i.e. the protonated form of 10; Scheme 3).
Hþ
Ka
h
i
h
i
k1 ¼ kneut
þ kprot
(1)
Ka þ Hþ
Ka þ Hþ
where kneut and kprot are the apparent first-order rate constants for
the decomposition of neutral and protonated forms of 5, Ka is the
acid dissociation constant of the protonated 5, and [Hþ]/(Ka þ [Hþ])
and Ka/(Ka þ [Hþ]) represent, respectively, the fraction of the
protonated and neutral forms of 5 present in solution. The kneut
values derived either from the pH-rate profiles or determined at pH
8 are presented in Table 1. The kinetically determined pKa values for
5a and 5s are 3.78 and 3.06, respectively, i.e. ca. 4 units lower that
those of Gly-Gly and Phe-Gly (pKa 8.2 and 7.6, respectively [34]). A
similar observation has been reported for imidazolidin-4-ones
derived from peptides [20].
3.2. Structural effects on chemical reactivity
The rate data presented in Table 1 show that the nature of the
substituents at C-2 of the imidazolidin-4-one moiety, and thus the
ketone starting material, also affects the reactivity of the neutral
form of the imidazolidin-4-ones 5. The order of reactivity (kneut) for
The imidazolidin-4-one moiety. Inspection of the kinetic data
presented in Table 1 reveals that the pH-independent pathway rate

N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
2509
the Phe-Gly–PQ imidazolidin-4-ones, according to the
ketone starting material, is cycloheptanone > cyclopentanone >
cyclohexanone > acetone, the cycloheptanone compound 5s being
ca. 20 times more reactive than its acetone counterpart, 5e. A
similar trend has been reported for imidazolidin-4-ones derived
from Gly–PQ, 3 [26], and from enkephalins, 11.[21] A good corre-
lation (r2 ¼ 0.99, Fig. 2) was observed between the log kneut values
for compounds 5e,n,q,r and those of their counterparts 11. This
strongly suggests that decomposition of 5 and 11 follow similar
pathways and present the same structural requirements. The
higher reactivity of the seven- and five-membered ring derivatives,
5s and 5r, when compared with the six- membered ring, 5n, can be
ascribed to a decrease in internal strain resulting from a change in
coordination number of the imidazolidin-4-one C-2 carbon atom
from four to three in the five-member ring-opening process. A
similar observation was reported for compounds 3. [26].
mosquitoes by interference with the cycle in these insects [35,36].
Compound 5f was the most potent compound at both dose levels,
reducing significantly (P < 0.05) the sporogonic development of
P. berghei when compared with the control. Notably, 5f was more
potent than primaquine, inhibiting almost completely the produc-
tion of oocysts at 50
mmol/kg. Compound 5b was the least active in
this screen. Adose of 10
mmol/kgof 5b did notsignificantly (P > 0.05)
affect oocyst production when compared with the control. This lack
of activity is unlikely to reflect the slow hydrolysis of 5b tothe parent
active dipeptide derivative, Ala-Gly–PQ (more than 99% of 5b is
converted to the active at the time of oocyst counting), but might
reflect the poor gametocytocidal activity of the parent dipeptide at
lower doses [18]. The decomposition of compounds 5a, 5b and 5f
was studied in plasma obtained from non-infected mice, diluted to
80% (v/v) with pH 7.4 isotonic phosphate buffer, at 37 ꢀC. These
compounds decomposed to corresponding dipeptidyl derivatives 9
The C-terminal amino acid. The effect of
a
-carbon atom substit-
in plasma, with first-order rate constants identical to those for the
kneut pathway (Table 1), indicating that plasma enzymes do not
catalyse the reaction.
uents (R1) in the C-terminal amino acid on the reactivity depends
on the nature of the R4 substituent at imidazolidin-4-one C-5
position. For example, the order of kneut for the Gly–AA1–PQ series
(i.e. R4 ¼ H) derived from cyclohexanone is Gly-Gly–PQ, 5i > Gly-
Phe–PQ, 5l > Gly-Leu–PQ, 5k z Gly-Ala–PQ, 5j, with 5i being only
ca. 4 times more reactive than 5j. This suggests that electron-
donating R1 substituents, although having only a minor influence
on the stability, retard the decomposition of imidazolidin-4-ones
5 in neutral and basic conditions (s* H ¼ 0.49; s* CH2Ph ¼ 0.21;
s* Me ¼ 0; s* Bui ¼ ꢁ0.12). In contrast, compound 5o, derived
from Ala-Ala–PQ and cyclohexanone, is ca. 7 times more reactive
than its Ala-Gly–PQ counterpart, 5m. Similarly, compound 5f,
derived from Ala-Ala–PQ and acetone, is ca. 15 times more reactive
than its Ala-Gly–PQ counterpart, 5b. This is consistent with the
finding that rate of decomposition of imidazolidin-4-ones derived
from dipeptides is accelerated when sterically-hindered amino
acids are present as C-terminal residues [20]. For example, the
imidazolidin-4-one derived from Phe-Ala and acetone is ca. 37
times more reactive than its Phe-Gly counterpart [20].
4. Conclusions
Primaquine dipeptide derivatives, 5, bearing an imidazolidin-4-
one moiety at the N-terminus are readily hydrolyzed to the parent
dipeptide derivatives of primaquine in neutral and basic solutions,
with half lives ranging from 0.7 to 31 h at 37 ꢀC, depending on the
substituents at the imidazolidin-4-one moiety as well as on the
C-terminal amino acid directly coupled to primaquine. The most
critical factor is the substituent at the imidazolidin-4-one C-2 position,
i.e. those derived from the ketone starting material. Imidazolidin-4-
ones derived from acetone are generally more stable than their
counterparts derived from cyclohexanone or cyclopentanone, while
the most reactive prodrug is the one derived from cycloheptanone.
Importantly, decomposition is not catalyzed by plasma enzymes.
Compounds 5arecapableofreducingthetransmissionof theinfection
to mosquitoes as efficiently as primaquine and the corresponding
parent dipeptide derivatives. In conclusion, these results herein pre-
sented suggest that imidazolidin-4-ones 5 present a stability profile
similartothatofimidazolidin-4-onesderivedfromdi-andtripeptides,
and thus may be a useful prodrug approach to protect the N-terminal
amino acid residue against enzymatic cleavage by aminopeptidases.
3.3. Antimalarial activity
The potential of compounds 5a, 5b and 5f (i.e. half lives of 3, 16
and 1 h, respectively) to prevent the transmission of malaria was
studied using a model consisting of BalbC mice infected with P. ber-
ghei and Anopheles stephensi mosquitoes and compared to that of
primaquine. The antimalarial activity was assessed based on the
percentage of mosquitoes with oocysts and the mean number of
oocysts per infected mosquito (Table 3). Although this model cannot
specifically attribute the drug effect to either gametocytocidal
activity or sporontocidal activity, it can clearly show if compounds
are effective at interrupting the transmission of the infection to
5. Experimental protocols
5.1. General
Boc-amino acids were from Bachem, solvents were all of p.a.
quality from Merck and were dried over pre-activated 3 Å molec-
ular sieves prior to utilization. All remaining reactants were from

2510
N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
Table 1
R4
R3
N
O
R2
Yields of imidazolidin-4-ones 5 (from precursors 9) and pH-independent pathway
rate constant constants, kneut, for the decomposition of neutral prodrugs 5 at 37 ꢀC.
O
H
Series
Compound R1
R4
Yield, 102,
N
NH
R2
COCHR1HN
%
kneut/
hꢁ1
N
H
R4
23.5
R3
R1
R4
5a
5b
H
H
H
41.2
(22.0a)
O
Me
56.5 4.39
(3.98a)
A
10
O
5c
5d
5e
5f
H
H
H
Me
Pri
75.2
74.8
69.4
2.20
7.01
4.52
Scheme 3.
Bui
NH
Me
N
CH2Ph
Me
N
tetramethylsilane (TMS) as internal reference: 1H and 13C chemical
shifts are given in parts per million (ppm) and proton–proton
coupling constants in Hertz (Hz); 1H NMR peak multiplicity is
indicated by br s (broad singlet), s (singlet), d (doublet), dd (double
doublet), t (triplet), q (quartet), m (unresolved multiplet). Mass
spectra (MS) were acquired by either the matrix-assisted laser
desorption ionization – time-of-flight (MALDI-TOF) using either
anthracene or 2,5-dihydroxybenzoic acid (DHB) as adjuvant
matrices, or the electrospray ionization ion-trap (ESI-IT) tech-
niques, on a Bruker Biflex II or a Finningan LCQ DECA XP MAX
spectrometer, respectively.
38.0 68.2
(63.1a)
H
Me
R1
5g
5h
Me
Bui
(CH2)2SMe 62.0 23.4
H
21.3 ND
5i
H
H
H
H
73.2 38.6
37.0 10.3
37.9 13.1
46.1 26.0
R4
5j
5k
5l
Me
Bui
CH2Ph H
O
O
5m
5n
5o
5p
H
H
Me
Me
Me
CH2Ph
Me
87.9
80.3
8.06
7.39
NH
N
N
H
46.4 60.2
(CH2)2SMe 80.2 36.7
R1
5.2. Syntheses
R4
O
5.2.1. Synthesis of compounds 8a–f
The appropriate PQAA1 [23] (3.1 mmol) was suspended in DCM
(30 mL), TEA (4 eq.) was added and the mixture was stirred in an
ice-water bath for 30 min. After addition of the BocAA4OH (1.1 eq.)
and HOBt (1.2 eq.), the carbodiimide (DCCI, 1.2 eq.) was slowly
added to the mixture, witch was kept at 0 ꢀC for more 2 h. The
reaction was allowed to proceed at room temperature for 2 d with
periodic monitoring by TLC. A second stepwise addition of carbo-
diimide (1.2 eq.) was made, and the reaction prolonged for further
3 d. The solid phase formed was removed by suction filtration and
identified as the carbodiimide-derived urea (DCU). The filtrate was
evaporated to dryness and the residue dissolved in the minimum
amount of warm acetone; the resulting solution was stored over-
night at 4 ꢀC and the urea precipitated was again removed by
suction filtration. The filtrate was evaporated to dryness and the
residue submitted to column chromatography on silica-gel, using
DCM/acetone mixtures as eluents. Products were isolated as
yellow-orange oils in 50–79% yields and successfully characterized
by MS and NMR (see Supporting information).
O
NH
N
N
H
5q
H
CH2Ph
61.8
5.30
R1
Me
R4
O
O
NH
N
5r
H
CH2Ph
77.8 12.2
N
H
R1
5.2.2. Synthesis of compounds 9a–f
Compounds 8 were dissolved in a small volume of neat tri-
fluoroacetic acid (TFA, ca. 5 mL) and the deprotection reactions
allowed to proceed for 1–2 h at room temperature. Excess TFA was
neutralized by dropwise addition of aqueous Na2CO3 at 30% until
pH 9; the supernatant oily phase formed in this process was
extracted six times with 10 mL portions of chloroform and the
R4
O
O
NH
N
5s
H
CH2Ph
33.9 94.9
N
H
R1
Table 2
First-order rate constants, kobs, for the hydrolysis of 5s in acetate and 5a in phos-
phate buffers at 37 ꢀC, with ionic strength maintained at 0.5 mol dmꢁ3 by addition of
NaClO4.
a
First-order rate constant for the hydrolysis in 80% human plasma in pH 7.4
phosphate buffer at 37 ꢀC.
Compd
Buffer
[Buffer], mol dmꢁ3
pH
102 kobs, hꢁ1
5s
CH3CO2H
0.005
0.010
0.050
0.002
0.005
0.010
4.45
4.47
4.42
6.93
6.95
6.96
94.7
95.4
95.7
24.6
23.8
25.5
Sigma–Aldrich. Silica-gel 60 plates (Merck F254) were used for thin-
layer chromatography. Nuclear magnetic resonance (NMR) spectra
were acquired on a Bruker AMX 300 spectrometer from solutions of
the compounds in deuterated chloroform (CDCl3) having
5a
H2POꢁ4

N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
2511
0.4
0.0
J ¼ 1.39 Hz; 1H, Q4); 7.31 (dd, J ¼ 8.25 Hz, J ¼ 4.17 Hz; 1H, Q3); 6.60
(m, 1H, –(CH2)3–NH–CO–); 6.34 (d, J ¼ 2.38 Hz; 1H, Q5); 6.26 (d,
J ¼ 2.32 Hz; 1H, Q7); 5.98 (d, J ¼ 8,09 Hz, 1H, –NH–CH(CH3)–CH2–);
3.89 (s; 3H, CH3–O–); 3.77 (s; 2H, –NH–CO–CH2–N–); 3.65–3.55 (m;
1H, –NH–CH(CH3)–CH2–); 3.46 (s; 2H, –N–CO–CH2–NH–); 3.30–
3.22 (m; 2H, –(CH2)2–CH2–); 1.90 (bs; 1H, –CO–CH2–NH–); 1.72–
1.58 (m; 4H, –CH2–CH2–CH2–); 1.33 and 1.31 (s þ s; 6H, –CH2–NH–
C(CH3)2–N–); 1.29 (d, J ¼ 6.56 Hz; 3H, –NH–CH(CH3)–CH2–). dC
(CDCl3, 75 MHz) 174.66 (–NH–CO–CH2–); 168.74 (–N–CO–CH2–
NH–); 159.38–91.71 (ArC); 78.66 (–N–C(CH3)2–NH–); 55.22 (CH3–
O–); 48.02 (–CO–CH2–NH–); 47.74 (–NH–CO–CH2–); 44.79 (–NH–
CH(CH3)–CH2–); 39.42 (–(CH2)2–CH2–); 33.87 (–CH2–(CH2)2–);
26.13 (–CH2–CH2–CH2–); 25.85 (–N–C(CH3)2–NH–); 20.54 (–NH–
CH(CH3)–CH2–). C22H31N5O3 (413.2427 g molꢁ1): m/z (MHþ) ¼
414.2231.
-0.4
-0.8
-1.2
-1.6
-2.0
0
2
4
6
8
10
12
14
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-2,2,5-
trimethylimidazolidin-4-one, 5b. Yield 56%; dH (CDCl3, 300 MHz)
8.52 (dd, J ¼ 4.24 Hz, J ¼ 1.70 Hz; 1H, Q2); 7.92 (dd, J ¼ 8.28 Hz,
J ¼ 1.62 Hz; 1H, Q4); 7.30 (dd, J ¼ 8.27 Hz, J ¼ 4.20 Hz; 1H, Q3); 6.63
(m; 1H, –CH2–NH–CO–CH2–); 6.33 (d, J ¼ 2.43 Hz, 1H, Q5); 6.26 (d,
J ¼ 2.38 Hz; 1H, Q7); 5.99 (d, J ¼ 8.10 Hz; 1H, –NH–CH(CH3)–CH2–);
3.89 and 3.88 (d þ d, J ¼ 15.28 Hz, J ¼ 15.36 Hz; 1H, –NH–CO–CHH–
N–); 3.87 (s; 3H, CH3–O–); 3.68 and 3.66 (d þ d, J ¼ 15.37 Hz,
pH
Fig. 1. pH–rate profiles for the hydrolysis of the imidazolidin-4-ones 5a (-) and 5s
(:) in aqueous solutions at 37 ꢀC.
organic phases pooled, dried over anhydrous MgSO4 and evapo-
rated to dryness. Products 9 were thus isolated as yellow-orange
oils in 84–99% yields and successfully characterized by MS an NMR
(see Supporting information).
J ¼ 15.31 Hz; 1H, –NH–CO–CHH–N–); 3.65–3.53 (m; 2H,
–
CO–CH(CH3)–NH–C-, –NH–CH(CH3)–CH2–); 3.33–3.18 (m; 2H,
–(CH2)2–CH2–); 1.79 (m; 1H, –CO–CH(CH3)–NH–C–); 1.69–1.57 (m;
4H, –CH2–CH2–CH2–); 1.40 (d, J ¼ 3.90 Hz; 3H, –CO–CH(CH3)–NH–
C–); 1.34–1.27 (m; 9H, –NH–C(CH3)2–, –NH–CH(CH3)–CH2–). dC
(CDCl3, 75 MHz) 176.63 (–N–CO–CH(CH3)–); 168.92 (–NH–CO–
CH2–N–); 159.38–91.69 (ArC); 67.95 (–N–C(CH3)2–NH–); 55.22
(CH3–O–); 53.65 (–CO–CH(CH3)–NH–); 47.72 (–NH–CH(CH3)–
CH2–CH2–); 45.14 (–CO–CH2–N–); 39.41 (–(CH2)2–CH2–); 33.97
5.2.3. Preparation of the target compounds 5a–s
Compounds 9 were mixed with an excess (2 eq.) of the appro-
priate ketone (propanone, cyclohexanone, cycloheptanone and 4-
methylciclohexanone) and TEA (1 eq.) in dry methanol (20 mL) and
the mixture refluxed for 3 d in the presence of 4 Å molecular sieves
(1 g). The reaction was monitored by TLC and ketone was re-added
(1 eq.) once per day. The molecular sieves were removed by
decantation and the solution evaporated to dryness. The oily
residue was submitted to column chromatography on silica-gel,
eluted with appropriate solvents. Fractions containing the chro-
matographically homogeneous product were pooled and evapo-
rated to dryness, yielding 5a–s as yellow-orange oils (21–88%) that
were analyzed by MS and NMR, as detailed below.
(–CH2–(CH2)2–);
27.71
(–CH2–CH2–CH2–);
26.23
(–N–
C(CH3)(CH3)–NH–); 25.37 (–N–C(CH3)(CH3)–NH–); 20.58 (–NH–
CH(CH3)–CH2–);
17.25
(–CO–CH(CH3)–NH–).
C23H33N5O3
(427.2583 g molꢁ1): m/z (MHþ) ¼ 428.2367.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-5-iso-
propyl-2,2-dimethylimidazolidin-4-one, 5c. Yield 75%; dH (CDCl3,
300 MHz) 8.46 (dd, J ¼ 4.09 Hz, J ¼ 1.31 Hz; 1H, Q2); 7.85 (dd,
J ¼ 8.22 Hz, J ¼ 1.32 Hz; 1H, Q4); 7.24 (dd, J ¼ 8.23 Hz, J ¼ 4.22 Hz;
1H, Q3); 6.66 and 6.64 (bs þ bs; 1H, –(CH2)3–NH–); 6.27
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-2,2-
dimethylimidazolidin-4-one, 5a. Yield 41%; dH (CDCl3, 300 MHz) 8.53
(dd, J ¼ 4.13 Hz, J ¼ 1.58 Hz; 1H, Q2); 7.92 (dd, J ¼ 8.26 Hz,
R4
O
R4
O
O
O
Ka
+
+
H+
N
NH2
N
NH
N
H
N
H
R1
R2
R3
R1
R2
R3
+
5, S
5, SH
kneut
kprot
R4
O
R2
R3
NH
N
H
NH2
+
O
R1
O
9
Scheme 4.

2512
N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
-2.4
-2.5
-2.6
-2.7
-2.8
-2.9
-3
300 MHz) 8.45 (dd, J ¼ 4.09 Hz, J ¼ 1.31 Hz; 1H, Q2); 7.86 (dd,
J ¼ 8.23 Hz, J ¼ 1.28 Hz; 1H, Q4); 7.24 (dd, J ¼ 8.27 Hz, J ¼ 4.26 Hz;
1H, Q3); 6.69 (bs; 1H, –(CH2)3–NH–); 6.27 (d, J ¼ 2.37 Hz; 1H, Q5;
6.21 (d, J ¼ 2.01 Hz; 1H, Q7); 5.94 and 5.93 (d þ d, J ¼ 7.85 Hz,
J ¼ 7.91 Hz; 1H, –NH–CH(CH3)–); 3.86 (d, J ¼ 15.62 Hz; 1H, –CO–
CHH–); 3.82 (s; 3H, CH3–O–); 3.63 and 3.61 (d þ d, J ¼ 15.47 Hz,
J ¼ 15.48 Hz; 1H, –CO–CHH–); 3.56–3.53 (m; 1H, –CH(NH–)–CH2–);
3.48–3.45 (m; 1H, –NH–CH(CH3)–); 3.24–3.12 (m; 2H, –(CH2)2–
CH2–); 1.77–1.68 (m; 3H, –CH2–CH(CH3)2); 1.64–1.54 (m; 4H, –CH2–
CH2–CH2–); 1.35 and 1.34 (s þ s; 3H, –C(CH3)(CH3)–); 1.24 and 1.23
(s þ s; 3H, –C(CH3)(CH3)–); 1.23 (d, J ¼ 5.55 Hz; 3H, –NH–CH(CH3)–
); 0.88 (d, J ¼ 6.23 Hz; 3H, –CH(CH3)(CH3)); 0.86 (d, J ¼ 6.13 Hz; 3H,
–CH(CH3)(CH3)). dC (CDCl3, 75 MHz) 176.50 and 176.48 (–CO–
CH(CH2–)–); 168.70 and 168.68 (–NH–CO–); 159.16 (QC6); 144.67
(QC2); 144.09 (QC8); 135.05 (QC4); 134.61 (QC10); 129.68 (QC9);
121.67 (QC3); 96.52 (QC7); 91.47 (QC5); 76.56 (–CH(NH–)–CH2–);
56.07 and 56.06 (–C(CH3)2–); 55.00 (CH3–O–); 47.53 (–NH–
CH(CH3)–); 44.70 and 44.67 (–CO–CH2–); 41.25 (–CH2–CH(CH3)2);
39.20 and 39.15 (–(CH2)2–CH2–); 33.79 and 33.77 (–CH2–CH2)2–);
30.73 (–CH(CH3)2); 27.51 and 27.50 (–C(CH3)(CH3)–); 26.03 and
26.01 (–C(CH3)(CH3)–); 24.98 (–CH2–CH2–CH2–); 23.10 and 23.08
(–CH(CH3)(CH3)); 21.30 (–NH–CH(CH3)–); 20.38 and 20.33
y = 1.5168x + 1.5983
R2 = 0.9951
-3.1
-3.2
-3.3
-3.4
-3.2
-3.1
-3
-2.9
-2.8
-2.7
-2.6
log kneut (5)
Fig. 2. Correlation of decomposition rates, kneut, of imidazolidin-4-ones 5e,n,q,r with
those of their enkephalin counterparts 11.
(–CH(CH3)(CH3)).
C26H39N5O3
(469.31 g molꢁ1):
m/z
(MHþ) ¼ 470.33; m/z (MNaþ) ¼ 492.47.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-5-benzyl-
2,2-dimethylimidazolidin-4-one, 5e. Yield 69%; dH (CDCl3, 300 MHz)
8.52 (dd, J ¼ 4.19 Hz, J ¼ 1.44 Hz; 1H, Q2); 7.92 and 7.91 (dd þ dd,
J ¼ 8.28 Hz, J ¼ 1.37 Hz and J ¼ 8.27 Hz, J ¼ 1.37 Hz; 1H, Q4); 7.32–7.16
(m; 6H, Q3, Ar2–6); 6.33 (d, J ¼ 2.64 Hz; 1H, Q5); 6.27 (d, J ¼ 2.45 Hz;
1H, Q7); 6.08–6.01 (m; 2H, –(CH2)3–NH–CO–, –NH–CH(CH3)–CH2–);
3.99 and 3.98 (d þ d, J ¼ 15.87 Hz and J ¼ 15.88 Hz; 1H, –NH–CO–
CHH–); 3.88 (s; 3H, CH3–O–); 3.86–3.83 (m; 1H, –CO–CH(CH2)–); 3.61
(m; 1H, –NH–CH(CH3)–CH2–); 3.52 and 3.51 (d þ d, J ¼ 15.88 Hz,
J ¼ 15.89 Hz; 1H, –NH–CO–CHH–); 3.19–3.00 (m; 4H, –(CH2)2–CH2–,
–CO–CH(CH2)–); 1.98–1.87 (m; 1H, –CO–CH–NH–); 1.67–1.51 (m; 4H,
–CH2–CH2–CH2–); 1.31 and 1.30 (s þ s; 3H, –N–C(CH3)(CH3)–NH–);
1.29 and 1.28 (s þ s; 3H, –N–C(CH3)(CH3)–NH–); 1.12 (d, J ¼ 5.92 Hz;
3H, –NH–C(CH3)–). dC (CDCl3, 75 MHz) 174.85 (–N–CO–CH–); 168.17
(–NH–CO–CH2–); 159.42–91.73 (ArC); 67.96 (–CO–CH(CH2)–NH–);
58.72 (–N–C(CH3)2–NH–); 55.20 (CH3–O–); 47.71 (–NH–CH(CH3)–
CH2–); 44.64 (–NH–CO–CH2–N–); 39.36 (–(CH2)2–CH2–); 36.33 (–CO–
CH(CH2)–); 33.96 (–CH2–(CH2)2–); 27.32 (–N–C(CH3)–CH3–NH–);
26.31 (–N–C(CH3)–CH3–NH–); 25.75 (–CH2–CH2–CH2–); 20.58 (–NH–
CH(CH3)–CH2–). C29H37N5O3 (503.2896 g molꢁ1): m/z (MHþ) ¼
504.2633.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-methyl-2-oxooc-
tyl}-2,2,5-trimethylimidazolidin-4-one, 5f. Yield 38%; dH (CDCl3,
300 MHz) 8.52 (dd, J ¼ 4.22 Hz, J ¼ 1.76 Hz; 1H, Q2); 7.91 (dd,
J ¼ 8.27 Hz, J ¼ 1.64 Hz; 1H, Q4); 7.71 (m; 1H, –(CH2)3–NH–CO–);
7.30 and 7.29 (dd þ dd, J ¼ 8.30 Hz, J ¼ 4.16 Hz and J ¼ 8.27 Hz,
J ¼ 4.20 Hz; 1H, Q3); 6.33 and 6.27 (d þ d, J ¼ 2.29 Hz and
J ¼ 2.42 Hz; 2H, Q5, Q7); 6.00 (m; 1H, –NH–CH(CH3)–); 3.88 (s;
3H, CH3–O–); 3.78–3.72 (m; 1H, –CO–CH(CH3)–); 3.64–3.49 (m;
2H, –CO–CH(CH3)–NH-, –NH–CH(CH3)–CH2–); 3.38–3.12 (m; 2H,
–CH2–CH2–CH2–); 1.72–1.61 (m; 8H, –CH2–CH2–CH2–, –CO–
CH(CH3)–NH–, –CO–CH(CH3)–N–); 1.40–1.26 (m; 12H, –N–
C(CH3)2–NH–CH(CH3)–CO-, –NH–CH(CH3)–CH2–). dC (CDCl3,
75 MHz) 176.85 (–N–CO–CH(CH3)–NH–); 172.34 (–NH–CO–
CH(CH3)–); 159.40–91.67 (ArC); 55.64 (–N–C(CH3)2–NH–); 55.22
(CH3–O–); 53.78 (–CO–CH(CH3)–NH–); 50.57 (–NH–CO–CH(CH3)–
); 47.75 (–NH–CH(CH3)–CH2–); 39.34 (–CH2–CH2–CH2–); 33.97
(–CH2–CH2–CH2–); 30.36 (–N–C(CH3)2–NH–); 26.17 (–CH2–CH2–
CH2–); 20.59 (–NH–CH(CH3)–CH2–); 16.84 (–CO–CH(CH3)–NH–);
15.54 (–NH–CO–CH(CH3)–). C24H35N5O3 (441.2740 g molꢁ1): m/z
(MHþ) ¼ 442.2897.
(d, J ¼ 2.31 Hz; 1H, Q5; 6.21 (d, J ¼ 2.20 Hz; 1H, Q7); 5.94 (d,
J ¼ 8.28 Hz; 1H, –NH–CH(CH3)–); 3.89 and 3.88 (d þ d, J ¼ 15.47 Hz,
J ¼ 15.45 Hz; 1H, –CO–CHH–); 3.82 (s; 3H, CH3–O–); 3.58(3) and
3.57(9) (d þ d, J ¼ 15.61 Hz, J ¼ 15.53 Hz; 1H, –CO–CHH–); 3.55–
3.52 (m; 1H, –CO–CH(CH–)–NH–); 3.44–3.42 (m; 1H, –NH–
CH(CH3)–); 3.27–3.12 (m; 2H, –(CH2)2–CH2–); 2.09–2.05 (m; 1H,
–CO–CH(CH–)–NH–); 1.64–1.49 (m; 4H, –CH2–CH2–CH2–); 1.33 (s;
3H, –C(CH3)(CH3)–); 1.22(5) (d, J ¼ 6.39 Hz; 3H, –NH–CH(CH3)–);
1.22(2) (s; 3H, –C(CH3)(CH3)–); 0.96 and 0.95 (d þ d, J ¼ 6.74 Hz,
J ¼ 6.80 Hz; 3H, –CH(CH3)(CH3)); 0.84 and 0.81 (d þ d, J ¼ 6.82 Hz,
J ¼ 6.84 Hz; 3H, –CH(CH3)(CH3)). dC (CDCl3, 75 MHz) 174.83 (–CO–
CH(CH–)–NH–); 168.72 (–NH–CO–CH2–); 159.16 (QC6); 144.67
(QC2); 144.05 (QC8); 135.05 (QC4); 134.58 (QC10); 129.66 (QC9);
121.65 (QC3); 96.50 (QC7); 91.43 (QC5); 76.02 and 76.01
(–CO–CH(CH–)–NH–); 62.50 (–C(CH3)2–); 54.98 (CH3–O–); 47.51
(–NH–CH(CH3)–); 44.45 (–CO–CH2–); 39.19 (–(CH2)2–CH2–); 33.77
(–CH2–CH2)2–); 30.72 (–CH(CH3)2); 28.95 (–C(CH3)(CH3)–); 25.57
and 27.56 (–C(CH3)(CH3)–); 26.10 and 26.05 (–CH2–CH2–CH2–);
20.34 (–NH–CH(CH3)–); 18.97 and 18.95 (–CH(CH3)(CH3)); 16.67
(–CH(CH3)(CH3)).
C25H37N5O3
(455.29 g molꢁ1):
m/z
(MHþ) ¼ 456.40; m/z (MNaþ) ¼ 478.40.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-5-iso-
butyl-2,2-dimethylimidazolidin-4-one, 5d. Yield 75%; dH (CDCl3,
Table 3
Effect of imidazolidin-4-ones 5a, 5b, 5f and primaquine on the sporogonic devel-
opment of Plasmodium berghei ANKA in Anopheles stephensi mosquitoes. Counting of
oocysts was carried out at day 10 post-feed.
Compound
Dose,
m
mol kgꢁ1
Mean no. of oocysts
% Of infected
mosquitoes
per mosquitoes, ꢂSEMa
1
10
50
10
50
10
50
10
50
0
12.2 (2.9)
0.2 (0.7)
4.8 (1.3)
3.8 (1.3)
81.3 (3.5)b
2.1 (0.2)
1.3 (0.2)
0.05 (0.1)
86.7 (4.0)
14.1
0.2
5.6
4.4
94.2
2.4
1.5
0.06
100
5a
5b
5f
Control
a
Mean standard error.
P > 0.05 versus control, by Student’s t test.
b

N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
2513
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-methyl-2-oxooc-
tyl}-5-[(2-methylthio)ethyl]-2,2-dimethylimidazolidin-4-one, 5g.
26.59 (–CH2– CH2– CH2–NH–); 25.05 (–NH–CH(CH3)–CH2–); 22.85
(–CH2–); 20.96 (–CH2–). C25H35N5O3 (453.2740 g molꢁ1): m/z
(MHþ) ¼ 454.3169; m/z (MNaþ) ¼ 476.2732.
Yield 62%; dH (CDCl3, 300 MHz) 8.50(4) and 8.50(9) (dd þ dd,
J ¼ 4.21 Hz, J ¼ 1.41 Hz and J ¼ 4.16 Hz, J ¼ 1.41 Hz; 1H, Q2); 7.89
(dd, J ¼ 8.26 Hz, J ¼ 1.60 Hz); 1H, Q4); 7.66–7.58 (m; 1H, –(CH2)3–
NH–); 7.28 (dd, J ¼ 8.27 Hz, J ¼ 4.20 Hz; 1H, Q3); 6.31 (d, J ¼ 2.42 Hz;
1H, Q5); 6.25 and 6.24 (d þ d, J ¼ 3.01 Hz and J ¼ 2.82 Hz; 1H, Q7);
5.98 (d, J ¼ 6.91 Hz; 1H, –NH–CH(CH3)–CH2–); 3.86(5) and 3.86(1)
(s þ s; 3H, CH3–O–); 3.76–3.72 (m; 1H, –CO–CH(CH3)–); 3.68–3.58
(m; 2H, –NH–CH(CH3)–CH2–, –CO–CH(CH2–Ph)–); 3.36–3.26 (m;
1H, –(CH2)2–CHH–); 3.21–3.10 (m; 1H, –(CH2)2–CHH–); 2.61–2.51
(m; 2H, –CH2–CH2–S–); 2.04 and 2.01 (s þ s; 3H, –CH2–S–CH3); 1.98
(bs; 1H, –CO–CH(CH2–)–NH–); 1.85–1.65 (m; 4H, –CH2–CH2–CH2–);
1.63 and 1.60 (s þ s; 6H, –N–C(CH3)2–NH–); 1.37 and 1.31 (bs þ bs;
3H, –CO–CH(CH3)–); 1.28(1) and 1.27(8) (d þ d, J ¼ 6.32 Hz,
J ¼ 6.34 Hz; 3H, –NH–CH(CH3)–CH2–). dC (CDCl3, 75 MHz) 176.13
and 176.07 (–CO–CH(CH2–)–NH–); 172.59 and 172.25 (–NH–CO–
CH(CH3)–); 159.81 (QC6); 145.33 (QC2); 144.73 (QC8); 135.76 and
135.73 (QC4); 135.19 (QC10); 130.32 and 130.30 (QC9); 122.28
(QC3); 97.12 and 97.07 (QC7); 92.08 (QC5); 78.21 (–CH(CH2–)–NH–
); 57.04 and 56.96 (–N–C(CH3)2–NH–); 55.79 and 55.72 (–CO–
CH(CH3)–); 55.64 and 55.63 (CH3–O–); 48.20 and 48.16 (–NH–
CH(CH3)–CH2–); 39.77 and 39.72 (–(CH2)2–CH2–); 34.39 (–CH2–
(CH2)2–); 31.02 and 31.01 (–CH2–CH2–S–); 30.79 and 30.76 (–CH2–
CH2–S–); 28.80 and 28.73 (–C(CH3)(CH3)–NH–); 26.66 and
26.64 (–CH2–CH2–CH2–); 26.11 and 26.03 (–C(CH3)(CH3)–NH–);
20.95 and 20.91 (–NH–CH(CH3)–CH2–); 16.02 (–S–CH3); 15.64
and 15.62 (–CO–CH(CH3)–). C26H39N5O3S (501.28 g molꢁ1): m/z
(MNaþ) ¼ 524.93.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-methyl-2-oxooc-
tyl}-1,4-diazaspiro[4.5]decan-2-one, 5j. Yield 37%; dH (CDCl3,
300 MHz) 8.52 (dd, J ¼ 4.16 Hz, J ¼ 1.42 Hz; 1H, Q2); 7.92 (dd,
J ¼ 8.26 Hz, J ¼ 1.57 Hz; 1H, Q4); 7.30 (dd, J ¼ 8.21 Hz, J ¼ 4.20 Hz;
1H, Q3); 6.72–6.59 (m; 1H, –(CH2)3–NH–); 6.33 (d, J ¼ 2.40 Hz; 1H,
Q5); 6.26 (d, J ¼ 2.35 Hz; 1H, Q7); 5.99 (bs; 1H, –NH–CH(CH3)–CH2–
); 3.89 (bs; 4H, CH3–O-, –CO–CH(CH3)–); 3.63–3.60 (m; 1H, –NH–
CH(CH3)–CH2–); 3.53–3.36 (m; 2H, –CO–CH2–NH–); 3.30–3.13 (m;
2H, –(CH2)–CH2–NH–); 2.34 (t, J ¼ 6.47 Hz; 1H, –CO–CH2–NH–);
1.90–1.79 (m; 4H, –CH2–CH2–CH2–NH–); 1.72–1.61 (m; 10H,
–(CH2)5–); 1.31 (d, J ¼ 6.97 Hz; 3H, –CO–CH(CH3)–); 1.28 (d,
J ¼ 6.38 Hz; 3H, –NH–CH(CH3)–). dC (CDCl3, 75 MHz) 177.49 (–NH–
CO–CH(CH3)–); 169.68 (–CO–CH2–NH–); 159.84–92.10 (ArC); 79.12
(–C–); 55.63 (CH3–O–); 53.80 (–CO–CH2–NH–); 48.81 (–CO–
CH(CH3)–); 48.14 (–NH–CH(CH3)–CH2–); 39.71 (–CH2)2–CH2–NH–);
37.66 and 36.12 (–CH2–); 33.53 (–CH2–(CH2)2–NH–); 27.44 (–CH2)2–
); 25.02 (–CH2–CH2–CH2–NH–); 23.23 and 22.53 (–CH2–); 21.00
(–NH–CH(CH3)–CH2–);
18.14
(–CO–CH(CH3)–).
C26H37N5O3
(467.2896 g molꢁ1): m/z (MHþ) ¼ 468.2452; m/z (MNaþ) ¼
490.2249.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-isobutyl-2-oxooctyl}-
1,4-diazaspiro[4.5]decan-2-one, 5k. Yield 38%; dH (CDCl3, 300 MHz) 8.52
(dd, J ¼ 4.22 Hz, J ¼ 1.63 Hz; 1H, Q2); 7.92 (dd, J ¼ 8.26 Hz, J ¼ 1.53 Hz;
1H, Q4); 7.30 (dd, J ¼ 8.25 Hz, J ¼ 4.20 Hz; 1H, Q3); 6.69 (bs, 1H,
–(CH2)3–NH–); 6.33 (d, J ¼ 2.51 Hz; 1H, Q5); 6.26 (d, J ¼ 2.46 Hz; 1H,
Q7); 5.98 (d, J ¼ 7.55 Hz; 1H, –NH–CH(CH3)–); 3.93–3.89 (m þ s; 4H,
–CH–CH2–CH(CH3)2, CH3–O–); 3.72–3.54 (m; 2H, –CO–CH2–NH–);
3.51–3.46 (m; 1H, –NH–CH(CH3)–CH2–); 3.30–3.20 (m; 2H, –(CH2)2–
CH2–NH–); 2.34 (t, J ¼ 6.52 Hz; 1H, –CO–CH2–NH–); 1.69–1.62 (m;
17H, –CH2–CH2–CH2–NH-, –CH–CH2–CH(CH3)2, –(CH2)5–); 1.29 (d,
J ¼ 6.29 Hz; 3H, –NH–CH(CH3)–); 0.94 and 0.92 (d þ d, J ¼ 5.41 Hz,
J ¼ 5.15 Hz; 6H, –CH–CH2–CH(CH3)2). dC (CDCl3, 75 MHz) 169.77 (–NH–
CO–CH(CH2–)–); 169.73 (–CO–CH2–NH–); 159.84–92.12 (ArC); 79.33
(–N(CO–)–C–); 56.53 (–CO–CH2–NH–); 55.65 (CH3–O–); 48.20 (–NH–
CH(CH3)–CH2–); 45.30 (–CO–CH(CH2–)–); 42.28 (–(CH2)2–CH2–NH–);
37.65 (–CH2–CH(CH3)2); 34.46 (–CH2–(CH2)2–NH–); 34.02 (–CH2–);
33.99 (–CH2–); 26.74 (–CH2–); 25.78 (–CH2–CH(CH3)2); 25.11 (–CH2–
CH2–CH2–NH–); 23.53 (–CH2–CH(CH3)(CH3)); 23.15 (–CH2–
CH(CH3)(CH3)); 22.62 (–CH2–); 22.13 (–NH–CH(CH3)–CH2–); 20.99
(–CH2–). C29H43N5O3 (509.3366 g molꢁ1): m/z (MHþ) ¼ 510.2777.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-benzyl-2-oxooc-
tyl}-1,4-diazaspiro[4.5]decan-2-one, 5l. Yield 46%; dH (CDCl3,
300 MHz) 8.54–8.50 (m; 1H, Q2); 8.02–7.93 (m; 1H, –(CH2)3–NH–
); 7.91 (dd, J ¼ 8.25 Hz, J ¼ 1.41 Hz; 1H, Q4); 7.33–7.17 (m; 6H, Q3,
Ar2–6); 6.32 (bs; 1H, Q5); 6.28 (bs; 1H, Q7); 6.02 (d, J ¼ 8.12 Hz;
1H, –NH–CH(CH3)–CH2–); 3.87 and 3.86 (s þ s; 3H, CH3–O–); 3.76–
3.73 (m; 1H, –CH–CH2–Ar); 3.67–3.60 (m; 1H, –NH–CH(CH3)–
CH2–); 3.36 and 3.34 (d; J ¼ 6,00 Hz, 2H, –CO–CH2–NH–); 3.30–
2.98 (m; 4H, –CH–CH2–Ar, –(CH2)2–CH2–NH–); 2.34 (t, J ¼ 6,44 Hz;
1H, –CO–CH2–NH–); 1.91–1.65 (m; 14H, –CH2–CH2–CH2–NH-,
–(CH2)5–); 1.31 (d, J ¼ 6.26 Hz; 3H, –NH–CH(CH3)–). dC (CDCl3,
75 MHz) 176.14 (–(CH2)3–NH–CO–); 172.58 (–N–CO–CH2–NH–);
159.84–92.10 (ArC); 82.53 (–C–); 63.11 (–CO–CH2–NH–); 55.62
(CH3–O–); 48.91 (–CH–CH2–Ar); 48.10 (–NH–CH(CH3)–CH2–);
39.76 (–CH2)2–CH2–NH–); 35.72 and 35.29 (–CH2–); 34.66 (–CH2–
(CH2)2–NH–); 33.57 (–CH–CH2–Ar); 26.65 (–CH2–); 24.90 (–CH2–
CH2–CH2–NH–); 22.98 and 22.70 (–CH2–); 20.96 (–NH–CH(CH3)–
CH2–). C32H41N5O3 (543.3209 g molꢁ1): m/z (MHþ) ¼ 544.2916.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-3-methyl-
1,4-diazaspiro[4.5]decan-2-one, 5m. Yield 88%; dH (CDCl3, 300 MHz)
8.52 (dd, J ¼ 4.22 Hz, J ¼ 1.65 Hz; 1H, Q2); 7.92 (dd, J ¼ 8.27 Hz,
J ¼ 1.55 Hz; 1H, Q4); 7.30 (dd, J ¼ 8.22 Hz, J ¼ 4.25 Hz; 1H, Q3); 6.68 (d,
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-isobutyl-2-oxooctyl}-
2,2-dimethylimidazolidin-4-one, 5h. Yield 21%; dH (CDCl3, 300 MHz)
8.54–8.52 (m; 1H, Q2); 7.92 (d, J ¼ 8.12 Hz; 1H, Q4); 7.32–7.28 (m; 1H,
Q3); 6.33 (d, J ¼ 1.69 Hz; 1H, Q5); 6.27 (d, J ¼ 1.58 Hz; 1H, Q7); 6.02–
5.92 (m; 1H, –NH–CH(CH3)–CH2–); 4.42–4.30 (m; 1H, –(CH2)3–NH–);
3.89 (s; 3H, CH3–O–); 3.89–3.86 (m; 1H, –CH(CO–)–CH2–); 3.66–3.53
(m; 2H, –NH–CH(CH3)–CH2–, –CH2–CH(CH3)2); 3.30–3.20 (m; 2H,
–(CH2)2–CH2–); 1.71–1.54 (m; 9H, –CH2–CH2–CH2–, –CO–CH2–NH-,
–CH2–CH(CH3)2); 1.30–1.13 (m; 9H, –N(CO–)–C(CH3)2, –NH–CH(CH3)–
CH2–); 0.94–0.81 (m; 6H, –CH2–CH(CH3)2). dC (CDCl3, 75 MHz) 170.67
(–CH2–NH–CO–); 166.40 (–CO–CH2–NH–); 159.81 (QC6); 145.32
(QC2); 144.74 (QC8); 135.77 and 135.72 (QC4); 135.29 and 135.25
(QC10); 130.34 (QC9); 122.31 (QC3); 97.26 (QC7); 92.18 (QC5); 67.20
(–N(CO–)–C(CH3)2–); 55.63 (CH3–O–); 52.73 (–CO–CH2–NH–); 48.25
(–NH–CH(CH3)–CH2–); 48.18 (–CH(CO–)(CH2–)–); 39.94 (–(CH2)2–
CH2–); 39.80 (–CH2–CH(CH3)2); 34.34 (–CH2–(CH2)2–); 30.72 and
30.64 (–C(CH3)(CH3)–NH–); 30.12 and 30.08 (–C(CH3)(CH3)–NH–);
25.34 (–CH2–CH2–CH2–); 25.19 (–CH2–CH(CH3)2); 21.93 (–CH2–
CH(CH3)(CH3)); 21.71 (–CH2–CH(CH3)(CH3); 20.96 (–NH–CH(CH3)–
CH2–). C26H39N5O3 (469.3053 g molꢁ1): m/z (MHþ) ¼ 470.2928.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-1,4-
diazaspiro[4.5]decan-2-one, 5i. Yield 73%; dH (CDCl3, 300 MHz) 8.53
(dd, J ¼ 4.19 Hz, J ¼ 1.72 Hz; 1H, Q2); 7.92 (dd, J ¼ 8.28 Hz;
J ¼ 1.59 Hz; 1H, Q4); 7.31 (dd, J ¼ 8.17 Hz, J ¼ 4.24 Hz; 1H, Q3); 6.61
(t, J ¼ 5.47 Hz; 1H, –(CH2)3–NH–); 6.34 (d, J ¼ 2.47 Hz; 1H, Q5); 6.26
(d, J ¼ 2.44 Hz; 1H, Q7); 5.99 (d, J ¼ 8.00 Hz; 1H, –NH–CH(CH3)–
CH2–); 3.89 (s; 3H, CH3–O–); 3.78(1) and 3.77(5) (s þ s; 2H, –NH–
CO–CHH–N–); 3.66–3.56 (m; 1H, –NH–CH(CH3)–CH2–); 3.42 (d;
J ¼ 1.06 Hz; 2H, –CO–CH2–NH–) 3.30–3.21 (m; 2H, –(CH2)2–CH2–
NH–); 2.34 (t, J ¼ 6.52 Hz; 3H, –CO–CH2–NH–); 1.88–1.58 (m; 14H,
–CH2–CH2–CH2–NH–, –(CH2)5–); 1.29 (d, J ¼ 6.41 Hz; 3H, –NH–
CH(CH3)–CH2–). dC (CDCl3, 75 MHz) 175.44 (–NH–CO–CH2–);
169.48 (–CO–CH2–NH–); 159.81–92.11 (ArC); 81.08 (–N–C–Ar);
55.64 (CH3–O–); 48.15 (–NH–CH(CH3)–CH2–); 45.06 (–CO–CH2–
NH–); 42.40 (–NH–CO–CH2–N–); 39.76 (–(CH2)2–CH2–NH–); 34.96
(–CH2–); 34.80 (–CH2–); 34.65 (–CH2–(CH2)2–NH–); 27.44 (–CH2);

2514
N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
J ¼ 4.82 Hz; 1H, –(CH2)3–NH–); 6.33 (d, J ¼ 2.48 Hz; 1H, Q5); 6.26 (d,
J ¼ 2.43 Hz; 1H, Q7); 5.99 (bs; 1H, –NH–CH(CH3)–CH2–); 3.89 (bs; 4H,
CH3–O-, –NH–CO–CHH–); 3.68–3.60 (m; 2H, –NH–CO–CHH-, –NH–
CH(CH3)–); 3.51 (q, J ¼ 6.88 Hz; -CO–CH(CH3)–NH–); 3.31–3.16 (m;
2H, –(CH2)2–CH2–NH–); 1.83 (d, J ¼ 4.34 Hz; 1H, –CO–CH(CH3)–NH–);
1.71–1.57 (m; 10H; –(CH2)5–); 1.52–1.46 (m; 4H, –CH2–CH2–CH2–NH–
); 1.33 and 1.30 (d þ d; J ¼ 7.05 Hz, J ¼ 7.01 Hz; 3H, –CO–CH(CH3)–
NH–); 1.29 (d, J ¼ 6.35 Hz; 3H, –NH–CH(CH3)–CH2–). dC (CDCl3,
75 MHz) 177.48 (–N–CO–CH(CH3)–); 169.66 (–NH–CO–CH2–);
159.82–92.12 (ArC); 79.12 (–N–C–NH–); 55.64 (CH3–O–); 53.80 (–NH–
CH(CH3)–CH2–); 48.20 (–CO–CH(CH3)–NH–); 45.32 (–NH–CO–CH2–
N–); 42.40 (–CH(CH3)–(CH2)2–CH2–); 39.73 (–CH2–); 37.58 (–CH2–);
34.43 (–CH(CH3)–CH2–(CH2)2)–); 27.31 (–CH2–); 26.61 (–CH(CH3)–
CH2–CH2–CH2–); 24.95 (–NH–CH(CH3)–CH2–); 23.00 (–CH2–);
22.46 (–CH2–); 20.99 (–NH–CH(CH3)–CH2–); 18.10 and 18.07 (–CO–
CH(CH3)–NH–). C26H37N5O3 (467.2896 g molꢁ1): m/z (MHþ) ¼
468.2162; m/z (MNaþ) ¼ 490.1942.
and J ¼ 4.96 Hz; 1H, –(CH2)3–NH–); 7.27 (dd, J ¼ 8.25 Hz,
J ¼ 4.21 Hz; 1H, Q3); 6.30 (d, J ¼ 2.47 Hz; 1H, Q5); 6.24 (bs; 1H, Q7);
5.98 (d, J ¼ 7.92 Hz; 1H, –NH–CH(CH3)–CH2–); 3.85(7) 3.85(4)
(s þ s; 3H, CH3–O–); 3.74 (q, J ¼ 7.36 Hz; 1H, –CO–CH(CH3)–); 3.64–
3.54 (m; 2H, –NH–CH(CH3)–CH2–, –CO–CH(CH2–)–NH–); 3.34–
3.26 (m; 1H, –(CH2)2–CHH–); 3.17–3.09 (m; 1H, –(CH2)2–CHH–);
2.59 and 2.56 (t þ t, J ¼ 7.30 Hz, J ¼ 7.02 Hz; 2H, –CH2–CH2–S–CH3);
2.33–2.28 (m; 2H, –CH2–CH2–S–); 2.04–2.01 (s þ s; 3H, –(CH2)2–S–
CH3); 1.87–1.79 (m; 5H, –CH2–CH2–CH2–, –CO–CH(CH2–)–NH–);
1.72–1.56 (m; 10H, –(CH2)5–); 1.61 (d, J ¼ 7.39 Hz; 3H, –CO–
CH(CH3)–); 1.27 (d, J ¼ 6.35 Hz; 3H, –NH–CH(CH3)–). dC (CDCl3,
75 MHz) 176.52 and 176.49 (–CO–CH(CH2)–NH–); 172.84 (–NH–
CO–CH(CH3)–); 159.84 and 159.82 (QC6); 144.33 (QC2); 144.70
(QC8); 135.75 and 135.73 (QC4); 135.16 (QC10); 130.30 (QC9);
122.25 (QC3); 97.08 and 97.06 (QC7); 92.06 (QC5); 80.16 (–CO–
CH(CH2–)–NH–); 56.92 and 56.87 (–N–C(–CH2–)–NH–); 55.63
(CH3–O–); 55.49 (–CO–CH(CH3)–); 48.20 and 48.14 (–NH–
CH(CH3)–CH2–); 39.73 (–(CH2)2–CH2–); 39.66 (–CH2–(CH2)4–);
37.84 and 37.82 (–(CH2)4–CH2–); 34.41 (–CH2–(CH2)2–); 34.05 and
34.00 (–(CH2)2–CH2–(CH2)2–); 31.47 (–CH2–CH2–S–); 30.76 and
30.73 (–CH2–CH2–S–); 26.70 and 26.64 (–CH2–CH2–CH2–); 23.35
(–(CH2)3–CH2–CH2–); 22.51 (–CH2–CH2–(CH2)3–); 20.92 and
20.91 (–NH–CH(CH3)–CH2–); 16.29 (–CH2–S–CH3); 15.67 (–CO–
CH(CH3)–). C29H43N5O3S (541.31 g molꢁ1): m/z (MHþ) ¼ 542.87;
m/z (MNaþ) ¼ 564.87.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-3-benzyl-
1,4-diazaspiro[4.5]decan-2-one, 5n. Yield 80%; dH (CDCl3, 300 MHz)
8.50 (dd, J ¼ 4.18 Hz, J ¼ 1.41 Hz; 1H, Q2); 7.90–7.89 (dd þ dd,
J ¼ 8.00 Hz, J ¼ 1.60 Hz and J ¼ 8.22 Hz, J ¼ 1.59 Hz; 1H, Q4); 7.30–
7.21 (m; 6H, Q3, Ar2–6); 6.31 (d, J ¼ 2.15 Hz; 1H, Q5); 6.26 (d,
J ¼ 2.31 Hz; 1H, Q7); 6.05–5.97 (m; 2H, –NH–CH(CH3)–CH2–
(CH2)2–NH–); 4.00–3.95 (m; 1H, –NH–CO–CHH–); 3.86 (s; 3H, CH3–
O–); 3.61–3.49 (m; 2H, –NH–CH(CH3)–CH2–, –CH(NH–)–CH2–Ar);
3.52(2) and 3.51(6) (d þ d, J ¼ 15.91 Hz, J ¼ 16.01 Hz; 1H, –NH–CO–
CHH–); 3.19–3.09 (m þ d, J ¼ 5.31 Hz, 4H, –CH2–Ar, –(CH2)2–CH2–);
3.01 (d, J ¼ 4.57 Hz; 1H, –CH(NH–)–CH2–Ar); 1.86–1.83 (m; 1H,
–CO–CH(CH2–Ar)–NH–); 1.74–1.68 (m; 2H, –CH2–CH2–CH2–NH–);
1.65–1.44 (m; 12H, –CH2–CH2–CH2–NH–, –(CH2)5–); 1.28 and 1.27
(d þ d, J ¼ 6.38 Hz, J ¼ 6.34 Hz; 3H, –NH–CH(CH3)–). dC (CDCl3,
75 MHz) 174.96 (–N(CO–)–); 168.59 (–NH–CO–CH2–); 159.27–91.54
(ArC); 78.47 (–CO–CH(CH2–Ph)–NH–); 58.43 (–N(CO–)–C–); 55.05
(CH3–O–); 47.58 (–NH–CH(CH3)–CH2–); 44.26 (–CO–CH2–N(CO–
)–); 41.84 (–(CH2)2–CH2–NH–); 39.16 (–CH2–Ph); 36.60
(–CH2–(CH2)2–NH–); 33.81 (–CH2–); 33.39 (–CH2–); 26.94 (–CH2–
CH2–CH2–); 26.15 (–CH2–); 22.51 (–CH2–); 21.94 (–CH2–); 20.44
(–NH–CH(CH3)–). C32H41N5O3 (543.3209 g molꢁ1): m/z (MHþ) ¼
544.2916.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-3-benzyl-
1,4-diazaspiro-8-methyl[4.5]decan-2-one, 5q. Yield 62%; dH (CDCl3,
300 MHz) 8.52 (dd, J ¼ 4.21 Hz, J ¼ 1.61 Hz; 1H, Q2); 7.90 and 7.89
(dd þ dd, J ¼ 8.26 Hz, J ¼ 1.77 Hz and J ¼ 8.28 Hz, J ¼ 1.78 Hz; 1H, Q4);
7.31–7.20 (m; 6H, Q3, Ar2–6); 6.32 (d, J ¼ 2.04 Hz; 1H, Q5); 6.26 (d,
J ¼ 2.44 Hz; 1H, Q7); 6.02–5.97 (m; 2H, –NH–CH(CH3)–CH2–, –NH–
CO–CH2–); 3.98 (d, J ¼ 15.96 Hz; 1H, –NH–CO–CHH–); 3.86 (s; 3H,
CH3–O–); 3.80 (q, J ¼ 4.87 Hz; 1H, –NH–CH(CH3)–CH2–); 3.62–3.57
(m; 1H, –CO–CH(CH2–)–NH–); 3.50 and 3.49 (d þ d, J ¼ 15.99 Hz,
J ¼ 16.01 Hz; 1H, –NH–CO–CHH–); 3.19–2.95 (m; 4H, –(CH2)2–CH2–,
–CH2–Ph); 2.00–1.39 (m; 14H, –CH2–CH2–CH2–, –CH(CH2–)–NH–,
–(CH2)2–CH(CH3)–(CH2)2–); 1.28(1) and 1.27(6) (d þ d, J ¼ 6.35 Hz
and J ¼ 6.38 Hz; 3H, –NH–CH(CH3)–); 0.86 and 0.85 (d þ d,
J ¼ 5.28 Hz and J ¼ 5.42 Hz; 3H, –(CH2)2–CH(CH3)–(CH2)2–). dC (CDCl3,
75 MHz) 175.55 (–CO–CH(CH2)–NH–); 169.12 (–NH–CO–CH2–);
159.85 (QC6); 145.36 (QC2); 144.75 and 144.72 (QC8); 139.98 (ArC1);
135.77 (QC4); 135.19 (QC10); 130.37 (ArC3 þ ArC5); 130.33 (QC9);
128.98 (ArC2 þ ArC6); 127.52 (ArC4); 122.30 (QC3); 97.10 (QC7);
92.11 and 92.09 (QC5); 78.79 (–CO–CH(CH2–)–NH–); 58.97 and
58.95 (–N(CO–)–C(–CH2–)–NH–); 55.62 (CH3–O–); 48.15 (–NH–
CH(CH3)–CH2–); 44.80 (–CO–CH2–N–); 39.72 (–(CH2)2–CH2–);
36.99 and 36.94 (–CH2–Ph); 34.43 and 34.39 (–(CH2)2–CH(CH3)–
(CH2)2–); 33.75 (–CH2–(CH2)2–); 31.63 (–CH2–CH2–CH(CH3)–
(CH2)2–); 31.52 (–(CH2)2–CH(CH3)–CH2–CH2–); 30.89 (–CH2–CH2–
CH(CH3)–(CH2)2–); 30.85 (–(CH2)2–CH(CH3)–CH2–CH2–); 26.72
(–CH2–CH2–CH2–); 22.23 (–NH–CH(CH3)–CH2–); 21.03 (–(CH2)2–CH
(CH3)–(CH2)2–). C33H43N5O3 (557.3366 g molꢁ1): m/z (Mþ) ¼
557.2001.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-3-benzyl-
1,4-diazaspiro[4.4]nonan-2-one, 5r. Yield 78%; dH (CDCl3, 300 MHz)
8.59 (dd, J ¼ 4.16 Hz, J ¼ 1.39 Hz; 1H, Q2); 7.99 (d, J ¼ 8.08 Hz; 1H,
Q4); 7.39–7.25 (m; 6H, Q3, –ArH); 6.40 (d, J ¼ 2.34 Hz; 1H, Q5); 6.34
(s; 1H, Q7); 6.10 (s; 1H, –(CH2)3–NH–); 6.02 (d, J ¼ 5.49 Hz; 1H,
–NH–CH(CH3)–); 4.11 (d, J ¼ 16.14 Hz; 1H, –CO–CHH–); 3.94(5) and
3.94(0) (s þ s; 3H, CH3–O–); 3.84 and 3.83 (t þ t, J ¼ 4.64 Hz,
J ¼ 4.66 Hz; 1H, –CO–CH(CH2–)–NH–); 3.66–3.62 (m; 1H, –NH–
CH(CH3)–); 3.50 (d; J ¼ 16.14 Hz; 1H, –CO–CHH–); 3.30–3.24 (m;
2H, –CH2–Ph); 3.16–3.04 (m; 2H, –(CH2)2–CH2–); 1.80–1.52 (m;
13H, –CH2–CH2–CH2–, –(CH2)4–, –CH(CH2)–NH–); 1.36 and 1.35
(d þ d, J ¼ 6.30 Hz, J ¼ 6.32 Hz; 3H, –NH–CH(CH3)–). dC (CDCl3,
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-methyl-2-oxooc-
tyl}-3-methyl-1,4-diazaspiro[4.5]decan-2-one, 5o. Yield 46%; dH
(CDCl3, 300 MHz) 8.49 (dd, J ¼ 4.18 Hz, J ¼ 1.56 Hz; 1H, Q2); 7.88
(dd, J ¼ 8.24 Hz, J ¼ 1.52 Hz; 1H, Q4); 7.77 (bs; 1H, –CH2–NH–CO–);
7.27 (dd, J ¼ 8.22 Hz, J ¼ 4.21 Hz; 1H, Q3); 6.30 (d, J ¼ 2.41 Hz; 1H,
Q5); 6.24 (bs; 1H, Q7); 5.99 (bs; 1H, –NH–CH(CH3)–); 3.86 (s; 3H,
CH3–O–); 3.73 (q, J ¼ 7.36 Hz; 1H, –NH–CO–CH(CH3)–); 3.59 (bs;
1H, –NH–CH(CH3)–CH2–);
3.47–3.44 (q þ q;
J ¼ 6.39 Hz,
J ¼ 6.71 Hz; 1H, –CO–CH(CH3)–NH–); 3.37–3.27 (m; 1H, –CH2–
CH2–CHH–); 3.20–3.09 (m; 1H, –CH2–CH2–CHH–); 1.89–1.77 (m;
1H, –CO–CH(CH3)–NH–); 1.70–1.59 (m; 14H, –(CH2)5–, –CH2–CH2–
CH2–NH–); 1.45–1.34 (m; 3H, –NH–CO–CH(CH3)–); 1.27 (d,
J ¼ 6.57 Hz; 6H, –NH–CH(CH3)–, –CO–CH(CH3)–NH–). dC (CDCl3,
75 MHz) 177.75 (–NH–CO–CH(CH3)–); 173.04 (–CO–CH(CH3)–NH–
); 159.82–92.07 (ArC); 80.13 (–N(CO–)–C–); 55.80 (CH3–O–); 55.62
(–CO–CH(CH3)–NH–); 54.06 (–NH–CH(CH3)–CH2–); 48.12 (–NH–
CO–CH(CH3)–); 39.66 (–(CH2)2–CH2–NH–); 39.59 (–CH2–); 37.87
(–CH2–); 34.36 (–CH2–(CH2)2–NH–); 26.68 and 26,54 (–CH2–);
25.14 (–CH2–CH2–CH2–); 23.42 (–NH–CH(CH3)–CH2–); 20.96
(–CO–CH(CH3)–NH–); 17.80 (–CH2–); 17.68 (–CH2–); 16.16 (–CO–
CH(CH3)–). C27H39N5O3 (481.3053 g molꢁ1): m/z (Mþ) ¼ 481.3849.
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-1-methyl-2-oxooc-
tyl}-3-[(2-methylthio)ethyl]-1,4-diazaspiro[4.5] decan-2-one, 5p.
Yield 80%; dH (CDCl3, 300 MHz) 8.49(3) and 8.49(0) (dd þ dd,
J ¼ 4.21 Hz, J ¼ 1.46 Hz and J ¼ 4.14 Hz, J ¼ 1.41 Hz; 1H, Q2); 7.88
(dd, J ¼ 8.25 Hz, J ¼ 1.56 Hz; 1H, Q4); 7.70 and 7.68 (t þ t, J ¼ 5.16 Hz

N. Vale et al. / European Journal of Medicinal Chemistry 44 (2009) 2506–2516
2515
75 MHz) 175.29 (–CO–CH(CH2–)–NH–); 168.31 (–NH–CO–CH2–);
159.37 (QC6); 144.70 (QC2); 144.05 (QC8); 136.25 (ArC1); 135.14
(QC4); 134.97 (QC10); 129.87 (QC9); 129.77 (ArC3); 129.74 (ArC6);
128.57 (ArC2 þ ArC7); 127.08 (ArC4); 121.78 (QC3); 96.76 (–CO–
CH(CH2–)–); 91.58 (QC7); 86.14 and 86.13 (QC5); 58.77 and 58.76
(–N(CO–)–C(–CH2–)–); 55.14 (CH3–O–); 47.64 (–NH–CH(CH3)–);
44.32 (–NH–CO–CH2–); 39.20 (–(CH2)2–CH2–); 36.82 (–CH2–Ph);
35.71 and 35.69 (–CH2–(CH2)3–); 34.66 (–CH2–(CH2)2–); 33.82 and
33.80 (–(CH2)3–CH2–); 26.20 and 26.16 (–CH2–CH2–CH2–); 22.99
(–CH2–CH2–(CH2)2–); 22.97 (–(CH2)2–CH2–CH2–); 20.48 (–NH–
CH(CH3)–). C31H39N5O3 (529.31 g molꢁ1): m/z (MHþ) ¼ 530.60; m/z
(MNaþ) ¼ 552.48.
analyzed by the HPLC method described above for the presence of
substrate and products.
5.5. In vivo gametocytocidal activity
BalbC mice were infected by intraperitoneal inoculations of 107
erythrocytes parasitized with P. berghei ANKA. After 4 days, when
the presence of gametocytes and exflagellation was observed by
microscopic observation of Giemsa stained blood films, mice were
randomly separated into five different groups of six animals. Each
group was treated by intraperitoneal administration with one
single dose of each compound 5 and primaquine (10 and 50 mmol/
3-{7-[(6-Methoxyquinolin-8-yl)amino]-3-aza-2-oxooctyl}-3-benzyl-
1,4-diazaspiro[4.6]undecan-2-one, 5s. Yield 34%; dH (CDCl3, 300 MHz)
8.51 (d, J ¼ 3.85 Hz; 1H, Q2); 7.90 (d, J ¼ 8.21 Hz; 1H, Q4); 7.31–7.15 (m;
6H, Q3, Ar2–6); 6.32 (bs; 1H, Q5); 6.26 (bs; 1H, Q7); 6.08 (d, J ¼ 6.11 Hz;
1H, –NH–CH(CH3)–CH2–); 6.00 (t, J ¼ 5.86 Hz; 1H, –NH–CO–CH2–);
3.99 (d, J ¼ 15.86 Hz; 1H, –NH–CO–CHH–); 3.87 (s; 3H, CH3–O–); 3.79–
3.76 (m; 1H, –NH–CH(CH3)–CH2–); 3.62–3.58 (m; 1H, –CO–CH(CH2–
)–NH–); 3.58 (d, J ¼ 15.80 Hz; 1H, –NH–CO–CHH–); 3.27–2.96 (m; 4H,
–(CH2)2–CH2–, –CH2–Ph); 1.85 (bs; 1H, –CO–CH(CH2–)–NH–); 1.67–
1.40 (m; 16H, –CH2–CH2–CH2–, –(CH2)6–); 1.28 (d, J ¼ 6.32 Hz; 3H,
–NH–CH(CH3)–CH2–). dC (CDCl3, 75 MHz) 175.28 (–CO–CH(CH2)–NH–
); 169.09 (–NH–CO–CH2–); 159.85 (QC6); 145.37 and 145.35 (QC2);
144.74 and 144.72 (QC8); 136.87 (ArC1); 135.77 (QC4); 135.19 (QC10);
130.32 (QC9); 130.31 (ArC3); 130.27 (ArC5); 129.02 (ArC2); 129.01
(ArC6); 127.55 (ArC4); 122.29 (QC3); 97.11 (QC7); 92.09 (QC5); 82.28
(–CO–CH(CH2–)–NH–); 58.68 (–N–C(–CH2–)–NH–); 55.63 (CH3–O–);
48.14 (–NH–CH(CH3)–CH2–); 45.33 (–NH–CO–CH2–); 40.58 and 40.55
(–CH2–Ph); 39.75 and 39.74 (–(CH2)2–CH2–); 37.84 and 37.80 (–CH2–
(CH2)5–); 36.59 (–(CH2)5–CH2–); 34.46 (–CH2–(CH2)2–); 29.66 and
29.63 (–(CH2)3–CH2–(CH2)2–); 29.32 and 29.28 (–(CH2)2–CH2–(CH2)3–
); 24.78 (–CH2–CH2–CH2–); 22.71 and 22.69 (–CH2–CH2–(CH2)4–);
22.31 and 22.27 (–(CH2)4–CH2–CH2–); 21.04 (–NH–CH(CH3)–CH2–).
C33H43N5O3 (557.3366 g molꢁ1): m/z (Mþ) ¼ 557.2182.
kg in inoculation volumes of 0.1–0.2 mL; controls consisted of mice
given a PBS solution). Two hours after administration, mice were
anesthetized and placed on top of individual cages containing ca.
50 glucose-starved A. stephensi female mosquitoes, which were
allowed to feed for 2 h. After the blood meal, unfed female
mosquitoes were removed from each cage. Ten days after the blood
meal, 10 mosquitoes of each cage were randomly collected and
dissected for microscopic detection of oocysts in midguts. For
further details see ref. [18].
Acknowledgments
The authors wish to thank FCT for financial support through the
research project PTDC/QUI/65142/2006, pluriannual funding to
iMed.UL, CIQUP and CMDT-LA, and a Post-Doctoral fellowship to
N.V. (SFRH/BPD/48345/2008).
Appendix. Supporting information
Supporting information associated with this article can be found
in the online version, at doi:10.1016/j.ejmech.2009.01.018.
References
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